The overall objective of this research is to develop and then use methods to measure intracellular oxygen concentrations in isolated cells in vitro, in tissue-like aggregates of cells (e.g. spheroids), and in tissues of intact animals. At the cellular and molecular level such information would be of value in the understanding of redox metabolism and cellular organization. At the clinical science level such information would be of value in detecting, characterizing and following any pathophysiological process that involves redox metabolism or altered oxygen concentrations. The processes noted above depend primarily on intracellular oxygen concentrations, but previously, due to technical difficulties, they have been studied primarily by measurements of extracellular oxygen concentrations or oxygen consumption. Our approach is to use the sensitivity and specificity of ESR and the effect of oxygen on ESR spectra (due to its effects on relaxation processes) to determine oxygen concentrations. In research carried out in the initial period of this grant, we used oxygen-sensitive nitroxides (plus extracellular paramagnetic agents to broaden away the spectra of extracellular nitroxides) and were able to measure intracellular oxygen concentrations in cell suspensions and show their relationship to the rate of oxygen consumption and to the rate of reduction of potential contrast agents for in vivo NMR studies. Full development of this methodology for cell suspensions and its application to physiological and pathophysiological conditions now seems feasible and warranted. Recent developments from several laboratories, including ours, now make it possible to extend the methodology to aggregates of cells such as multicellular spheroids and to whole animals using ESR and NMR imaging techniques. Our specific aims in the present proposal, in order of priority and emphasis, are: 1) To improve the accuracy, sensitivity and applicability of measurements of intracellular oxygen in suspensions of cells under baseline conditions and under various physiological and pathophysiological conditions; 2) To develop methods to measure intracellular oxygen concentrations at specific intracellular sites including membranes, cytoplasm, lysosomes, mitochondria, and nuclei; 3) To extend the measurements of intracellular oxygen to models of tissues, such as spheroids and tissue slices, under various conditions; 4) To extend the measurement of intracellular and extracellular oxygen concentrations to tissues of intact animals; and 5) To explore the feasibility of measuring the intracellular concentration of singlet oxygen.